Host cells inhibit a broad range of microbes by producing nitric oxide (NO.). The goal of this project is to understand how NO. inhibits bacteria and how pathogenic bacteria resist NO.. Our hypothesis is that host-derived NO. interacts with bacterial metal- and thiol-containing targets to produce fundamental metabolic alterations that prevent bacterial growth. The specific aims of this proposal are to answer important mechanistic questions by analyzing specific novel NO. targets: [1] How do bacteria sense and respond to host-derived NO.?-- NsrR, a nitrite- sensitive repressor of defenses against nitrosative stress;[2] How does NO. constrain bacterial metabolism?-- LpdA, or lipoamide dehydrogenase, an essential subunit of enzymes required for the TCA cycle and amino acid biosynthesis [3] How does NO. disrupt redox homeostasis?-- PFL, or pyruvate-formate lyase, which converts pyruvate to acetyl CoA and formate to facilitate the regeneration of NAD+ under oxygen-limited conditions NO. cytotoxicity will be analyzed in two important bacterial pathogens. NsrR and LpdA will be studied in the enteric bacterium Salmonella Typhimurium. PFL will be investigated in the gram-positive bacterium Staphylococcus aureus, which employs a unique strategy to bypass PFL inhibition by NO.. This project will examine a variety of potential molecular mechanisms for protein inactivation by NO through destabilization of iron-sulfur clusters, iron-nitrosyl formation, S-nitrosylation, or glycyl radical quenching. The aims will be achieved by a combination of genetic, biochemical and in vivo analyses in routine use in our laboratory. Insights from these studies will provide an integrated picture of how host- derived NO. alters the metabolism of invading bacteria to impair growth and how pathogens can overcome these actions by detoxifying NO. or bypassing crucial sites of inhibition. PROJECT NARRATIVE The body's immune cells produce nitric oxide to limit the growth of microorganisms. This research project analyzes the mechanisms by which nitric oxide exerts toxic effects on bacteria and the mechanisms by which pathogenic bacteria such as Salmonella and Staphylococcus resist this important host defense mechanism. Such studies will help to identify novel strategies for the prevention or treatment of infection.